The present invention relates to methods of immortalizing primary cells and to the immortalized cell lines produced by these methods.
The desirability of generating immortalized cell lines for use as in vitro models of various tissues has long been recognized. For example, researchers have endeavored for some time to generate a suitable in vitro model for cells of the anterior pituitary gland. The anterior pituitary gland of mammals is composed of specialized cells known to synthesize and secrete a variety of hormones that regulate critical body functions. Corticotropes express adrenocorticotropic hormone (ACTH) which regulates the steroid hormone output from the adrenal gland. Gonadotropes express the gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which operate in concert to control reproductive functions. Thyrotropes are the source of thyroid-stimulating hormone (TSH), which regulates the synthesis and release of thyroid hormones from the thyroid gland. Somatotropes express growth hormone (GH), which controls cell growth. Lactotropes express a hormone closely related to growth hormone, prolactin which controls lactation and other functions as well. Prolactin was discovered in 1970 and its entire functional role is not yet understood (Hodson, Calif., 1996). Melanotropes express melanocyte-stimulating hormone (MSH), which regulates pigmentation of the skin. The suffix xe2x80x9ctrophxe2x80x9d may be substituted for xe2x80x9ctropexe2x80x9d when referring to these cells. Control of end-organs regulated by anterior pituitary hormones is closely regulated by circulating end-organ hormones and neural inputs which interact at pituitary and hypothalamic sites to maintain homeostasis.
Some pituitary cell lines presently exist but these are usually neoplastic cells which continue to divide in vitro by virtue of malignant transformation, e.g., the GH-3 rat cell line was derived from a pituitary tumor. GH-3 cells are termed somatomammotrophs because they synthesize and secrete both prolactin and growth hormone. Other rat pituitary hormone-secreting cell lines are also known, e.g., GH-1, which was derived from the same tumor as the GH-3 line. The RC-4B/C cell line is an apparent mix of corticotropes, thyrotropes, lactotropes and somatotropes. Likewise, with regard to human pituitary cell lines, most of these come from neoplasms of the pituitary. Prolactinoma is the most prevalent form of pituitary neoplasm in man. Therefore, human lactotrope adenoma cells have been extensively studied to determine the genetic mechanisms of tumor formation; such studies suggest that expression of the hst gene encoding fibroblast growth factor may be associated with pituitary tumorigenesis (Cai, W Y, et.al., 1994; Gonsky, R et.al., 1991). Hydridomas formed by fusion of malignant cells with gonadotropes have been used to express human FSH (U.S. Pat. No. 4,383,034). LH has also been produced by fusion of human pituitary adenoma cells with a human lymphoblastoid cell line (U.S. Pat. No. 4,383,035).
Transformed cells are not the same as non-transformed cells present in the body. Transformation shifts the expression of endogenous proteins significantly, turning off the expression of some proteins, while the expression of other proteins is increased. Transformation also alters morphological and cellular properties, e.g. transformed cells require lower amounts of serum and growth factors to support proliferation. Transformed cells may also erroneously process proteins resulting in molecular alteration of endogenous proteins. Human cytomegalovirus immediate-early promoter was more efficient in directing reporter gene expression when expressed in transformed than non-transformed rat anterior pituitary cells suggesting that transformation alters transcriptional factors (Coleman, T A, et.al., 1991). Thus, a transformed pituitary cell may not reflect the normal physiological and cellular processes of its progeny.
Hormone-secreting cells of the pituitary can be grown in vitro as a primary culture. Such primary cultures of pituitary cells have been used extensively to investigate physiological processes involved in regulation of hormone secretion. For example, these cultures were used to bioassay specific hypothalamic releasing hormones that regulate hormone output of the pituitary. This discovery provided considerable insights into the mechanisms of intercommunications between the nervous and endocrine systems (Guillemin, 1978). However, after several cell divisions primary cultures invariably reach a crisis stage and thereafter cease to divide. This property is referred to as senescence. There are some pituitary cell lines which continue to proliferate in culture, but are apparently non-transformed. Mouse thyrotrope tumors such as TtT 97 result from thyroidectomy, synthesize and release TSH, and respond to T3 and TRH (Furth, J, 1955; Condliff, P G, et.al., 1969; Cacicedo, L, et.al., 1981). The rat somatomammotroph cell line, rPCO and clonal derivatives, was derived from primary culture of rat pituitary cells in media containing T3 and GHRH, which may have resulted in selective proliferation of somatotropes. RPCO cells synthesize and release growth hormone and prolactin; secretion is differentially affected by TRH, T3 and GHRH (Chomczynski, P, et.al., 1988; Kashio, Y, et.al., 1990). Both of these cell lines have been used in basic research into the molecular biology of pituitary hormone processing (Coleman, T A, et.al., 1991; Wood, W M, et.al., 1989). The mechanisms by which these cells are immortalized is unknown. These cells have not yet been definitively demonstrated to be non-transformed (Chomczynski, P, et.al., 1988).
Recombinant DNA technology has also been used to produce protein hormones of the pituitary gland. The non-glycosylated monomers of growth hormone and prolactin were initially expressed in E. coli cells using a recombinant plasmid containing the cDNA for growth hormone or prolactin. Recombinant protein accumulates in inclusion bodies primarily as reduced monomers. Following solubilization with urea, reoxidized monomers were recovered through use of reoxidation procedures which resulted in biologically active material similar to native hormones with the addition of a methionine residue at the amino terminus (Paris, N, et.al., 1990). The group of heterodimeric glycoprotein hormones including hCG, LH, FSH and TSH have also been expressed in Chinese Hamster Ovarian (CHO) cells using cloned sequences of both the homologous xcex1-subunit and the hormone-specific xcex2-subunit (Reddy, V B, et.al., 1985; Simon, J A, et.al., 1988; Keen, J L, et.al., 1989; U.S. Pat. No. 4,923,805; U.S. Pat. No. 5,156,957; U.S. Pat. No. 4,840,896). These heterodimeric proteins are known to be glycoproteins containing 15%-35% carbohydrate as N-linked and O-linked glycans present on both the xcex1 and xcex2-subunits. These carbohydrates add structural complexity and have various functions in the assembly, stabilization, modulation of biological activity and control of clearance of these molecules (Szkudkinski, M W, et.al., 1996; Galway, A B, et.al., 1990). The addition of carbohydrate moieties to the protein backbone is a function of the host cell and since these are different from the natural hormone-producing cells, variation in the glycosylation of recombinant hormones may result. Detailed comparison of urinary and recombinant human FSH (rhFSH) has shown that rhFSH is more acidic, suggesting differences in sialic acid content or terminal monosaccharides (de Leeuw, R, et.al., 1996). Also, recombinant human TSH (rhTSH) contains only sialic acid at terminal biantennary monosaccharides while pituitary-derived hTSH also contains sulfated N-acetyl galactosamine terminal residues. This results from the lack of N-acetyl galactosamine transferase in CHO cells used for the expression of rhTSH (Szkudlinski, M W, et.al., 1996).
There are non-transformed cells in the art which are thought to arise through spontaneous immortalization as occurs in non-malignant or benign tumors. However, these cell lines are relatively rare. Some other cells are known to undergo spontaneous immortalization without transformation. Studies of such cells showed that c-myc was over expressed while expression of other establishment oncogenes was unaltered, possibly due to a chromosomal translocation (Tavassoli and Shall, 1988; Madsen, et.al., 1992). Transfection of the v-myc oncogene into a murine macrophage cell line resulted in immortalization, suggesting that over expression of the myc oncogene itself was sufficient to induce immortality. Furthermore, the biological properties of the immortalized cell line were similar to normal cells (Blasi, et al., 1987). A variety of other studies have also demonstrated the immortalization effect of v-myc transfection into several different types of cells, including those with secretory functions common to endocrine cells (Strom, et.al., 1991; Bernard, et.al., 1989; Briers, et.al., 1993; Vanderstichele, et.al., 1994; Briers et.al., 1994; Hoeben, et.al., 1995).
Expression of the cellular myc gene is normally repressed by Lef/Tcf transcription factors which bind to specific sequences of the c-myc promoter (He, T C, et.al., 1998). In some forms of cancer and normal development, c-myc expression is increased through disruption of the developmental pathway known in the art as Wnt. The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is mutated in about 85% of all human colon cancers. The APC gene product is a component of the Wnt pathway that normally complexes xcex2-catenin, preventing its effect on the nucleus. Mutations of the APC gene or activation of the Wnt pathway via ligand-receptor interaction result in disruption of the APC-xcex2-catenin complex allowing xcex2-catenin access to the nucleus. xcex2-catenin then binds to Tcf transcription factors, de-represses the c-myc gene resulting in its increased transcription (He, TC, et.al., 1998; Dale, 1998). Increased cell proliferation in response to developmental signals and in colon cancer is mediated by increased c-myc expression.
The protein product of the myc gene is a transcription factor which forms a heterodimeric complex with another protein, Max. Myc is a central regulator of cell proliferation through transcription effects, including repression and activation of target gene expression, e.g., MrDd and cdc25A (George, K H; 1996). The latter target also induces apoptosis in cells. Max also forms homodimers and heterodimers with Mad and Mxi-1, alternative partners to Myc, which compete with Myc/Max for common gene targets. These various interactions are thought to regulate the ultimate effect of Myc over expression, i.e., cell differentiation, immortalization without transformation, transformation, or cell death (Facchini, L M and Penn, L Z; 1998; Desbarats, L, et.al., 1996; Amati, B and Land, H; 1994).
The SV40 virus has been used to immortalize several cells of animal and human origin. The pSV3neo plasmid containing the complete SV40 early genetic region including the large T and small T antigens was used to immortalize rat Leydig cells (Nagpal, M L, et.al., 1994). Two cell lines resulted which were maintained for 35 passages without apparent transformation as indicated by an absence of the anchorage-independent growth in soft agar. The large T antigen coding sequence had become integrated into the cellular genome. These cells displayed many characteristics of differentiated Leydig cells including expression of LH receptors (LH-R), insulin-like growth factor I (IGF-I) and IGF-I receptors (IGF-IR) and IGF binding protein 2 (IGFBP-2). The amounts of transcripts of the LH-R gene were lower, IGF-I, IGF-IR were the same and IGFBP-2 were much higher in the immortalized cells. Also, the immortalized cells could not synthesize testosterone due to low levels of the enzyme P450scc. Hence, the immortalized cells maintained some, but not all, differentiated characteristics of rat Leydig cells (Nagpal, M L, et.al., 1994). In order to avoid production of virus particles in permissive and semipermissive cells, including human and monkey cells, SV40 plasmids containing the large T antigen gene were modified to block viral replication by deleting part of the viral origin of replication, the so-called orin SV40 mutant (Gluzman, 1980). Such mutants have been particularly valuable in the immortalization of human cells (Chow, J Y, 1989), including granulosa cells, fetal liver epithelial cells, breast and other epithelial cells (Byong-Lyul, L, et.al., 1996; Ishida, T, et.al., 1995; Berthon, et.al., 1992; Lechner, M S and Laimins, L A, 1991). Human cell lines immortalized by SV40 Large T antigen exhibit increased growth in culture. However, they usually reach secondary senescence and are no longer viable (Stein, G H, 1985).
The degree of differentiation of the cells depends on the status of differentiation at the time of transfection. Thus, fetal liver cells immortalized by the plasmid pMK16-SV40(orixe2x88x92) failed to express xcex1-fetoprotein (AFP) possibly because it had not yet differentiated at the time of establishment of the cell line (Ishida, T, et.al., 1995). The SV40 large T antigen is thought to combine with the products of the tumor suppressor genes p53 and p105-Rb, neutralize anti-oncogenic effects of these genes, including activation of apoptosis and thereby increase cell proliferation. Such effects may also lead to malignant transformation. Two immortalized breast epithelial cell lines resulted from transfection by the SV40 large T antigen. One of these lines remained non-transformed while another resulted in a transformed cell line, showing that SV40 large T antigen can result in immortalization and malignant transformation (Berthon, P, et.al., 1992).
The temperature sensitive mutant of SV40 large T (tsA58) has also been used to immortalize cells, including human pituitary thyrotropes. At the permissive temperature, the large T antigen is expressed and cells exhibit a transformed phenotype. At the non-permissive temperature, large T antigen is no longer expressed and cells revert to the normal differentiated phenotype (Chou, J Y, 1989). Adult human thyrotropes transfected with tsA58 plasmid proliferated at the permissive temperature 33xc2x0 C. and showed no growth at 39xc2x0 C. These cells have undergone more than 150 passages. Contrary to adult thyrotropes, these immortalized thyrotropes did not express TRH receptors nor do these cells secrete hTSH (Ham, et.al., 1998). The large T antigen of SV40 has also been used to immortalize specific pituitary tumor cells at discrete stages of development by constructing large T antigen-containing transgenes driven by various promoters that are differentially activated during development (Windle, J J, et.al., 1990; Alarid, E T, et.al., 1996). A hybrid of the adenovirus-12 and the SV40 virus has also been used to immortalize human prostate cells. These cells express many of the normal phenotypic characteristics of human prostate cells and were non-tumorigenic (U.S. Pat. No. 5,610,043).
Accordingly, a need exists for methods of immortalizing primary cultures of various cell lines that are not transformed. The present invention satisfies this need and provides related advantages.
The present invention relates to novel methods for the generation of immortalized cell lines from primary cultured cells and to such immortalized cell lines. Such cell lines are immortalized lines derived, for example, from hormone-producing cells of the pituitary gland, i.e., lactotropes, somatotropes, thyrotropes, gonadotropes, corticotropes or melanotropes. Supporting cells normally associated with endocrine cells such as follicular stellate cells may also be immortalized as described herein as well as neurons, glial cells, corneal epithelial cells, and xcex2-islet cells of the pancreas which produce insulin.
Such cell lines are immortalized but not transformed as occurs during malignant transformation of a normal cell into a cancerous cell. Hence the immortalization of these cells is not secondary to malignant transformation.
Furthermore, the cells of a given line are maintained in culture under conditions allowing expression of the adult phenotype of the cell. For pituitary cells, the adult phenotype includes properties such as: presence of the appropriate hormone and secretory ultrastructure, secretion of such hormone in response to those secretagogues which normally regulate hormone secretion in the adult organism, receptors for the hypothalamic releasing hormone normally present on adult cells, expression of hormone-encoding genes, and expression of appropriate differentiation factors such as, for example, Pit-1 or SF-1 (Simmons, D M, et.al., 1990; Sanno, N, et.al., 1998; Bedford, et.al., 1996).
The present invention also refers to immortalization of cells from different species. Thus primary cells immortalized according to the present invention may be derived from various species, including for example, human, equine, bovine, canine, rat or mouse.
A feature of the present invention is that it involves use of both environmental factors and genetic manipulation of primary cultured cells to achieve or maintain immortalization without transformation at the fully differentiated phenotype. Environmental factors, such as the osmolarity of the cell culture media, addition of various growth factors and tissue extracts have been used to extend the longevity of cultured pituitary cells (e.g., U.S. Pat. Nos. 4,124,448 and 5,747,341). Significant effects of environmental factors on primary culture of pituitary cells, including effects on differentiation and proliferation have also been observed. In one embodiment, the present invention involves use of environmental factors to induce specific states of differentiation and/or proliferation prior to use of establishment oncogenes including, for example, the large T antigen of the SV40 virus and controlled expression of the myc proto-oncogene to immortalize cells. In another embodiment, specific environmental conditions can be used to maintain immortalized cell cultures that already possess desired properties such as a particular state of differentiation, hormone expression or proliferation without the need to induce such states. Furthermore, environmental conditions may also be used to control the expression and differentiation of cells that have been immortalized according to the methods of the present invention.
The present invention offers significant advantages over the prior art. This invention provides an alternative source of relatively pure native protein hormones for various applications in research, diagnostics and therapeutics. Presently, native material is derived through the extraction of tissues and the purification of each hormone present within such tissue. The physical and chemical properties of some hormones are closely related, for example pituitary growth hormone and prolactin are structurally similar. Prolactin is thought to have evolved from growth hormone (Cooke, N E, et.al., 1981). Thus, complete elimination of one hormone from the other is difficult; usually, purified hormone is at least partially contaminated with the other.
The present invention provides for cultures of pure hormone-producing cells without contamination by cells which produce other hormones. Thus, for example, this invention allows a pure culture of immortalized lactotropes without somatotropes. Likewise a clonal culture of pure somatotropes is possible without the presence of lactotropes. Hence, cultures of immortalized lactotropes provide a crude prolactin sample devoid of contamination by growth hormone and somatotrope cultures provide crude growth hormone uncontaminated by prolactin. Optimization of the secretion of the desired pituitary hormone provides additional advantages in purification to homogeneity as the hormone content in the culture media may be enriched, while other contaminating proteins are minimized. Purification methods well-known in the art (Sinha, Y N) may then be applied to culture media enriched by the secretion of, for example, prolactin or growth hormone exclusively to generate highly purified material uncontaminated by related hormones. Absolute purity of hormones is desirable in such applications as hormone structure-function determinations and therapeutic uses of these hormones. Gonadotropes are known to produce both LH and FSH. Hence, gonadotrope cultures do not provide sources of impure hormone devoid of gonadotropin contamination.
Another advantage of the present invention in its use as an alternative source for native pituitary hormones concerns the state of glycosylation of the heterodimeric glycoprotein hormones LH, FSH and TSH. Glycans are added post-translationally to the polypeptide chains composing the alpha and beta subunits. Glycosylation is a property of the cell expressing the hormone. As previously noted, rhTSH contains only sialic acid at terminal biantennary monosaccharides while pituitary-derived hTSH also contains sulfated N-acetyl galactosamine terminal residues resulting from the lack of N-acetyl galactosamine transferase in CHO cells used for the expression of rhTSH (Szkudlinski, M W, et.al., 1996). Since the present invention utilizes the endogenous cell from which native hormone arises, such a source can provide glycosylation patterns closer to native hormones than heterologous cells. The structure of the carbohydrate containing moieties contained in pituitary hormones is known to be an important determinant of function. A study comparing pituitary-derived human FSH, rhFSH, chemically deglycosylated FSH, and FSH expressed in a baculovirus expression vector/Sf9 cell system which fails to glycosylate FSH found substantial loss of biological activity in the deglycosylated forms of FSH. FSH deglycosylation is known to result in inhibitory intracellular events (Arey, et.al., 1997). Thus while the complete structure-function relations of FSH are not yet known, glycosylation is a key structural determinant of function. Hence, use of the present invention to produce large quantities of native glycoprotein hormones is likely to advance detailed studies of structure and function of these hormones. The present invention also provides another source of natural isoforms of native protein hormones for detailed study of their functional roles. Another advantage of the present invention is that cell lines or donors may be tested for viral contamination, including; HIV 1 and 2, HBV, HCV, etc prior to immortalizing the cells, thus providing a safe source of native pituitary hormones.
Additionally, the present invention allows investigation of the molecular biology of pituitary hormone producing cells in vitro without compromise of results due to cellular transformation. For example, determination of the molecular biology of the transforming growth factor family of proteins including activin, follistatin and inhibin is an appropriate application of the immortalized gonadotropes and other target cells. Many of the interactions between regulatory pathways may be altered by transformation. Since the cells of the present invention are not transformed, natural regulatory pathways are intact and suitable for direct investigation. The molecular biology of particular cells may be utilized to optimize expression of a given pituitary hormone and its secretion. Manipulation of these latter parameters effects the specific cellular expression of a given pituitary hormone. Higher levels of specific cellular expression are desirable methods to reduce the cost of the production of a therapeutic product such as cell line-derived FSH. Furthermore, cellular molecular biology may be utilized to extend the functional lifetime in culture, also resulting in a lower production costs.
A final advantage of the present invention concerns the ability to immortalize human pituitary hormone producing cells. While transgenic approaches have been used to immortalize rat pituitary cells at various stages of differentiation, such studies are not realistically applicable to humans because of ethical and regulatory issues regarding transgenic human beings. However, since the present invention utilizes primary cultured cells, including human pituitary cells, such cells may be immortalized and various embodiments of the present invention can be applied to immortalization at different stages of differentiation, given that these can be established in primary culture.